Human serum albumin (hereinafter referred to simply as HSA) is the most abundant protein contained in plasma. It contributes to the maintenance of osmotic pressure in blood and binds to nutrients and metabolites to thereby transport these substances. HSA having these functions has been employed as a drug for treating hypoalbuminemia caused by an albumin loss or reduction in albumin synthesis, and in hemorrhagic shock.
HSA has been produced mainly from a fraction of collected blood. However, the process for producing HSA from blood has such problems as sporadic supply of blood, economical disadvantage, and contamination with undesirable substances such as hepatitis virus. Thus there has been an urgent requirement to develop a material usable as a substitute for naturally occurring USA.
Under these circumstances, techniques for the mass production and purification of HSA by means of gene manipulation (as a substitute for the HSA originating in blood) have been developed as recombinant DNA technology has progressed
To purify the HSA obtained by gene manipulation (hereinafter referred to as recombinant HSA and abbreviated as rHSA), it is not appropriate to apply the conventional processes for purifying HSA originating in plasma as such. This is because the impurities to be eliminated from rHSA completely differ from those contained in the HSA originating in plasma. Namely, rHSA is contaminated with, for example, coloring matters characteristic to recombinant HSA, proteins originating in the host cells, polysaccharides, etc. In particular, it is necessary to sufficiently eliminate components originating in the host cells, since they are foreign matters for living organisms including human being and thus can cause a problem of antigenicity.
Accordingly, there have been carried out various studies in order to isolate and purify to a sufficient degree rHSA produced via culture from components originating in the host cells and culture components. One of the conventional processes is exemplified by the process which comprises subjecting a yeast culture medium containing rHSA to pressing--ultrafiltration membrane treatment--heating--ultrafiltration membrane treatment and then treating by procedures such as chromatography using a cation exchanger and an anion exchanger, and hydrophobic chromatography JP-A-5-317079 (the term "JP-A" as used herein means an "unexamined Japanese patent application") corresponding to EP-A-570916, Biotechnology of Blood Proteins, 1993, 227, 293-298!. Further, the process comprising the above-described procedure followed by the chelate resin treatment or the boric acid/borate treatment has been reported (EP-A-570916, JP-A-6-245789 corresponding to EP-A-612761).
In the above-mentioned conventional process, it is essentially required to effect the purification consisting of the above-mentioned several steps to thereby eliminate antigens originating in the host cells and achieve a high degree of purification. On the other hand, this process has such disadvantages as a decrease in the yield of rHSA and a prolonged treating period due to large number of steps. Although attempts have been made to elevate the yield of each step so as to improve the yield of rHSA, it seems that no further improvement in yield can be made and thus the yield of rHSA has already reached the upper limit. Moreover, the conventional process described above suffers from another problem that the pressing is effected in an open system and thus there is a risk of contamination. Namely, hygienic management, which is essentially required in the production of rHSA as a medicine, is highly difficult therein. In addition, the degree of coloring of rHSA can be reduced only to a A350/A280 ratio of about 0.015 (in the case of a solution containing 250 mg/ml of rHSA) at the lowest (JP-A-7-170993 and JP-A-7-170994 corresponding to EP-A-658569). on the other hand, there has been developed a process for recovering a target protein directly from a crudo culture medium without affecting any pre-treatment such as elimination of cells or concentration of the medium, after the completion of the cultivation (e.g., the streamline method with the use of expanded bed adsorption technique developed by Pharmacia, International Publication In Japan No. 6-500050 corresponding to EP-A-538467).
No case has been reported so far on the application of the above-mentioned expanded bed adsorption technique to the purification of rHSA, in particular, the recovery and purification of rHSA from a yeast culture medium. Thus, it remains unknown whether or not such a method is actually useful in the rationalization of the purification of rHSA and the improvement in the yield of the same. However, it is expected that the application of this method or one similar thereto to the purification of rHSA would contribute to the simplification of the conventional purification treatment consisting of several steps.
However, there arises a problem that, under the acidic conditions employed for the adsorption by a streamline column (adsorbent: Streamline SP) for use in the above-mentioned method, rHSA contained in the culture medium is rapidly degraded by proteases contained in the culture medium and thus the yield of rHSA is seriously lowered. It is, therefore, difficult to apply the above-mentioned expanded bed adsorption technique as such to the purification of rHSA.
Thus, there has boon an urgent requirement to develop a process by which rHSA can be highly purified in a stable state at a high yield without spoiling the merits of the expanded bed adsorption technique (i.e., the simplification and rationalization of the purification process, etc.).